PAK 2: modulators of lymphocyte activation

ABSTRACT

The present invention relates to regulation of lymphocyte activation. More particularly, the present invention is directed to nucleic acids encoding PAK2, which is involved in modulation of lymphocyte activation. The invention further relates to methods for identifying and using agents, including small organic molecules, peptides, circular peptides, antibodies, lipids, antisense nucleic acids, and ribozymes, that modulate lymphocyte activation via modulation of PAK2; as well as to the use of expression profiles and compositions in diagnosis and therapy related to lymphocyte activation and suppression.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Ser. No. 60/280,647,filed Mar. 30, 2001, herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to regulation of T lymphocyte activation.More particularly, the present invention is directed to nucleic acidsencoding PAK2, which is involved in modulation of T lymphocyteactivation and TCR signaling. The invention further relates to methodsfor identifying and using agents, including small organic molecules,peptides, circular peptides, antibodies, lipids, antisense nucleicacids, and ribozymes, that modulate lymphocyte activation and TCRsignaling via modulation of PAK2; as well as to the use of expressionprofiles and compositions in diagnosis and therapy related to lymphocyteactivation and suppression.

BACKGROUND OF THE INVENTION

The immune response includes both a cellular and a humoral response. Thecellular response is mediate largely by T lymphocytes (alternatively andequivalently referred to herein as T cells), while the humoral responseis mediated by B lymphocytes (alternatively and equivalently referred toherein as B cells). Lymphocytes play a number of crucial roles in immuneresponses, including direct killing of virus-infected cells, cytokineand antibody production, and facilitation of B cell responses.Lymphocytes are also involved in acute and chronic inflammatory disease;asthma; allergies; autoimmune diseases such as scleroderma, perniciousanemia, multiple sclerosis, myasthenia gravis, IDDM, rheumatoidarthritis, systemic lupus erythematosus, and Crohn's disease; and organand tissue transplant disease, e.g., graft vs. host disease.

B lymphocytes produce and secrete antibodies in response to theconcerted presentation of antigen and MHC class II molecules on thesurface of antigen presenting cells. Antigen presentation initiates Bcell activation through the B cell receptor (BCR) at the B cell surface.Signal transduction from the BCR leads to B cell activation and changesin B cell gene expression, physiology, and function, including secretionof antibodies.

T cells do not produce antibodies, but many subtypes of T cells produceco-stimulatory molecules that augment antibody production by B cellsduring the humoral immune response. In addition, many T cells engulf anddestroy cells or agents that are recognized by cell surface receptors.Engagement of the cell surface T cell receptor (TCR) initiates T cellactivation. Signal transduction from the TCR leads to T cell activationand changes in T cell gene expression, physiology, and function,including the secretion of cytokines.

Identifying ligands, receptors, and signaling proteins downstream ofTCR, as well as BCR, activation is important for developing therapeuticregents to inhibit immune response in inflammatory disease, autoimmunedisease, and organ transplant, as well as to activate immune response inimmunocompromised subjects, and in patients with infectious disease andcancer (see, e.g., Rogge et al., Nature Genetics 25:96-101 (2000); U.S.Pat. Nos. 5,518,911; 5,605,825; 5,698,428; 5,698,445; 6,013,464; and6,048,706).

SUMMARY OF THE INVENTION

The present invention provides nucleic acids encoding PAK2, which is aserine/threonine kinase involved in modulation of T lymphocyteactivation and TCR signaling. The invention therefore provides methodsof screening for compounds, e.g., small organic molecules, antibodies,peptides (such as PAK2 kinase domain fragments), circular peptides,lipids, antisense molecules, and ribozymes, that are capable ofmodulating lymphocyte activation, including TCR signaling, e.g., eitheractivating or inhibiting T lymphocytes. Therapeutic and diagnosticmethods and reagents are also provided.

In one aspect of the invention, nucleic acids encoding PAK2 areprovided. In another aspect, the present invention provides nucleicacids, such as probes, antisense oligonucleotides, and ribozymes, thathybridize to a gene encoding a PAK2 protein. In another aspect, theinvention provides expression vectors and host cells comprisingPAK2-encoding nucleic acids. In another aspect, the present inventionprovides PAK2 protein, and antibodies thereto.

In another aspect, the present invention provides a method foridentifying a compound that modulates T lymphocyte activation, themethod comprising the steps of: (i) contacting a T cell comprising aPAK2 polypeptide or fragment thereof with the compound, the PAK2polypeptide or fragment thereof encoded by a nucleic acid thathybridizes under stringent conditions to a nucleic acid encoding apolypeptide having an amino acid sequence of SEQ ID NO:2; and (ii)determining the chemical or phenotypic effect of the compound upon thecell comprising the PAK2 polypeptide or fragment thereof, therebyidentifying a compound that modulates T lymphocyte activation.

In another aspect, the present invention provides a method foridentifying a compound that modulates T lymphocyte activation, themethod comprising the steps of: (i) contacting the compound with a PAK2polypeptide or a fragment thereof, the PAK2 polypeptide or fragmentthereof encoded by a nucleic acid that hybridizes under stringentconditions to a nucleic acid encoding a polypeptide having an amino acidsequence of SEQ ID NO:2; (ii) determining the physical effect of thecompound upon the PAK2 polypeptide; and

(iii) determining the chemical or phenotypic effect of the compound upona cell comprising the PAK2 polypeptide or fragment thereof, therebyidentifying a compound that modulates T lymphocyte activation.

In one embodiment, the host cell is primary T lymphocyte or a cultured Tlymphocyte, e.g., a Jurkat cell.

In another embodiment, the chemical or phenotypic effect is determinedby measuring CD69 expression, NFAT expression, CD40L expression, IL-2production, intracellular Ca²⁺ mobilization, Ca²⁺ influx, or lymphocyteproliferation.

In another embodiment, modulation is inhibition of T lymphocyteactivation.

In another embodiment, the polypeptide is recombinant. In anotherembodiment, the PAK2 polypeptide comprises an amino acid sequence of SEQID NO:2. In another embodiment, the PAK2 polypeptide is encoded by anucleic acid comprising a nucleotide sequence of SEQ ID NO:1.

In another embodiment, the compound is an antibody, antisense molecule,small organic molecule, peptide, or a circular peptide.

In another aspect, the present invention provides a method of modulatingT lymphocyte activation in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of acompound identified using the methods described above.

In one embodiment, the subject is a human.

In another asepct, the present invention provides a method of modulatingT lymphocyte activation in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of aPAK2 polypeptide, the polypeptide encoded by a nucleic acid thathybridizes under stringent conditions to a nucleic acid encoding apolypeptide having an amino acid sequence of SEQ ID NO:2.

In another aspect, the present invention provides a method of modulatingT lymphocyte activation in a subject, the method comprising the step ofadministering to the subject a therapeutically effective amount of anucleic acid encoding a PAK2 polypeptide, wherein the nucleic acidhybridizes under stringent conditions to a nucleic acid encoding apolypeptide having an amino acid sequence of SEQ ID NO:2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of PAK proteins.

FIG. 2 shows an nucleotide (SEQ ID NO:1) and amino acid (SEQ ID NO:2)sequence for human PAK2.

FIG. 3 provides an alignment of PAK1, 2, and 3 amino acid sequences.

FIG. 4A shows phenotypic assays in Jurkat cells. FIG. 4B showsidentification of inhibitory hits.

FIG. 5A shows a diagram of target validation. FIG. 5B shows PAK2 mutantproteins identified in a CD69 assay.

FIG. 6A shows successful phenotype transfer of PAK2ΔS. FIG. 6B showssuccessful phenotype transfer of PAK2ΔL.

FIG. 7A shows cell specificity of the PAK2ΔL effect. FIG. 7B shows TCRinduced CD69 upregulation IRES-vector control.

FIG. 8A shows DN-Syk inhibits both TCR and BCR signaling. FIG. 8B showsthat PAK2ΔL specifically inhibits PCR signaling.

FIG. 9A shows TaqMan quantitative detection of PAK1 or PAK2 mRNA. FIG.9B shows the relative level of PAK1 message in various human tissues.

FIG. 10A shows the relative level of PAK2 mRNA in various human tissues.FIG. 10B shows the relative level of PAK2 mRNA in various human celllines and cell populations.

FIG. 11A shows retroviral infection of primary T lymphocytes. FIG. 11Bshows primary T cell assays.

FIG. 12A shows that anti-CD3 alone was not sufficient to induce IL-2secretion. FIG. 12B shows that PAK2ΔS inhibits anti-CD3/anti-CD28induced IL-2 secretion.

FIG. 13A shows that PAK2ΔL inhibits receptor-mediated IL-2 secretion inprimary T cells. FIG. 13B shows that PAK2ΔL inhibits receptor-mediatedCD40L up-regulation in primary T cells.

FIG. 14A shows wild type and kinase inactivated PAK2. FIG. 14B shows atransient overexpression assay to examine the TCR induced CD69upregulation.

FIG. 15A shows that PAK2ΔL inhibits TCR signaling. FIG. 15B shows thatwild-type PAK2 does not inhibit CD69.

FIG. 16A shows that kinase inactive PAK2 partially inhibits CD69. FIG.16B shows a summary of PAK2 inhibition of CD69.

FIG. 17A shows an assay for determining the involvement of PAK2 in TCRsignaling. FIG. 17B shows that TCR stimulates PAK2 kinase activity.

FIG. 18 shows the nucleotide sequence for PAK2ΔS and PAK2ΔL (SEQ ID NO:3and SEQ ID NO:4).

FIG. 19A shows PAK2 mRNA expression in tissues. FIG. 19B shows PAK2 mRNAexpression in primary lymphocytes.

FIG. 20A shows that PAK2ΔL inhibits calcium influx. FIG. 20B shows thevector control for calcium influx.

FIG. 21 shows that PAK2ΔL inhibits NFAT activation.

FIG. 22 shows that PAK2 kinase activity is required for TCR-induced NFATactivation.

FIG. 23A shows a model for a trans-dominant fragment directly inhibitingthe kinase domain. FIG. 23B shows generation of a kinase inhibitorysegment.

FIG. 24A shows the effect of GFP-PAK2 fragments on CD69. FIG. 24B showsthe effect of GFP-PAK2 fragments on Jurkat TAg CD69 (ratio of GFP).

FIG. 25A shows the effect of GFP-PAK2 fragments on Jurkat TAgCD69 (%inhibition). FIG. 25B shows binding of the PAK3 fragment to the kinasedomain.

FIG. 26 shows that the tranx-dominant GFP-PAK2 fragmentco-immunoprecipitates with the kinase domain.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

A protein from the PAK (“p21 activated kinase”) family has beenfunctionally identified as a protein involved in regulating T lymphocyteactivation and TCR signaling. PAK2 was identified in a functionalgenetic screen using CD69 as a readout of T cell activation. Nucleicacids encoding mutant variants of PAK2 (SEQ ID NOS:3-4) were recoveredas inhibitors of T cell activation-induced CD69 expression. Mutant PAK2expression in Jurkat cells results in inhibition of TCR induced CD69upregulation, calcium influx, and NFAT activatiaon. Mutant PAK2expression also inhibited receptor-mediated IL-2 production and CD40Lupregulation in human primary lymphocytes. Peptides and fragments of thekinase domain can be used to bind to and inhibit the kinase domain.These fragments inhibit TCR-induced NFAT activation and CD69 activation.The present application also demonstrates that PAK2 is involved in theTCR signaling pathway. These results indicate that PAK2 modulators canbe used for inhibition of TCR signaling and lymphocyte activation.

PAK family proteins (“p21-activating kinases”) are serine/threoninekinases of the ste20 subfamily that act as GTPase effectors, serving astargets for small GTP-binding proteins such as Cdc42 and Rac. PAK familyproteins also bind and/or phosphorylate histones H2B and H4, PIXs, MLCK,and paxillin. PAK family proteins have been implicated in a number ofbiological activities, including cytoskeletal reorganization and nuclearsignaling following stimulation of various receptors (see, e.g.,Bagrodia & Cerione, Trends Cell Biol. 9:350-355 (1999)). The familyincludes PAK1, PAK2, PAK3 and PAK4.

Human PAK2 protein has a molecular weight of approximately 58 kDa (525amino acids) and is encoded by a gene located on chromosome 3 (see,e.g., Martin et al., EMBO J. 14:1970-1978 (1995); Martin et al., EMBO J.14:4385 (1995); Manser et al., J. Biol. Chem. 270:25070-25078 (1995);and Knaus et al., Science 269:221-223 (1995)). PAK2 mRNA is ubiquitouslyexpressed and appears to be alternatively spliced, with transcripts of7.5 kb, 5 kb, 4.4 kb, and 3 kb detected in most tissues. Jurkat cellsexpress PAK2 protein, and in these cells, PAK2 protein is activated byproteolytic cleavage during caspase-mediated apoptosis (see, e.g., Rudel& Bokock, Science 276:1571-1574 (1997); Bokoch, Cell Death Differ.5:637-645 (1998)). PAK1 was previously implicated in TCR signaling (Kuet al., EMBO J. 20:457-465 (2001)). A highly conserved HIV protein, NEF,is specifically associated with PAK2 but not PAK1, and NEF is known tointerfere with CD3 signaling in T cells (Renkema et al., Curr. Biol.(2000), Renkema et al., J. Virol. (2001); Luria et al., Proc. Nat'lAcad. Sci USA 88:5326-5330 (1991)). Despite these features, thebiological function of PAK2 is not well understood.

The present invention identifies PAK2 as a member of the TCR signalingpathway. The present invention, therefore, has functionally identifiedPAK2 as drug targets for compounds that suppress or activate Tlymphocyte activation, preferably T lymphocyte activation, e.g., for thetreatment of diseases in which modulation of the immune response isdesired, e.g., for treating diseases related to T lymphocyte activation,such as delayed type hypersensitivity reactions; asthma; allergies;autoimmune diseases such as scleroderma, pernicious anemia, multiplesclerosis, myasthenia gravis, IDDM, rheumatoid arthritis, systemic lupuserythematosus, and Crohn's disease; and conditions related to organ andtissue transplant, such as graft vs. host disease; and acute and chronicinflammation; as well as in diseases in which activation of the immuneresponse is desired, e.g., in immunocompromised subjects, e.g., due toHIV infection or cancer; and in infectious disease caused by viral,fungal, protozoal, and bacterial infections. Preferably, modulators arecompounds that inhibit PAK2 and thereby inhibit T cell activation.

Definitions

By “disorder associated with T lymphocyte activation” or “diseaseassociated with lymphocyte activation” herein is meant a disease statewhich is marked by either an excess or a deficit of T cell activation,including TCR signaling. For example, lymphocyte activation disordersassociated with increased activation include, but are not limited to,acute and chronic inflammation, asthma, allergies, autoimmune diseaseand transplant rejection. Pathological states for which it may bedesirable to increase lymphocyte activation include HIV infection thatresults in immunocompromise, cancer, and infectious disease such asviral, fungal, protozoal, and bacterial infections.

The terms “PAK2” protein or fragment thereof, or a nucleic acid encoding“PAK2” or a fragment thereof refer to nucleic acids and polypeptidepolymorphic variants, alleles, mutants, and interspecies homologs that:(1) have an amino acid sequence that has greater than about 60% aminoacid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequenceidentity, preferably over a region of over a region of at least about25, 50, 100, 200, 500, 1000, or more amino acids, to an amino acidsequence encoded by a PAK2 nucleic acid (SEQ ID NO:1) or amino acidsequence of a PAK2 protein (SEQ ID NO:2); (2) specifically bind toantibodies, e.g., polyclonal antibodies, raised against an immunogencomprising an amino acid sequence of a PAK2 protein (SEQ IS NO:2),immunogenic fragments thereof, and conservatively modified variantsthereof; (3) specifically hybridize under stringent hybridizationconditions to an anti-sense strand corresponding to a nucleic acidsequence encoding a PAK2 protein (SEQ ID NO:1), and conservativelymodified variants thereof; (4) have a nucleic acid sequence that hasgreater than about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%,preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or highernucleotide sequence identity, preferably over a region of at least about25, 50, 100, 200, 500, 1000, or more nucleotides, to a PAK2 nucleic acid(SEQ ID NO:1).

A PAK2 polynucleotide or polypeptide sequence is typically from a mammalincluding, but not limited to, primate, e.g., human; rodent, e.g., rat,mouse, hamster; cow, pig, horse, sheep, or any mammal. The nucleic acidsand proteins of the invention include both naturally occurring orrecombinant molecules. Exemplary nucleic acid and protein sequences forhuman PAK2 are provided by GenBank Accession Nos. NM_(—)002577,NP_(—)002568.1, XM_(—)039354, U25975.1, and AF092132 (see also FIGS. 2and 18, which provide exemplary nucleotide and amino acid sequences forhuman PAK2). As described herein, PAK2 proteins have serine/threoninekinase activity, which can be assayed using standard methodology knownto those of skill in the art (see, e.g., Manser et al., J. Biol. Chem.270:25070-25078 (1995)).

The phrase “functional effects” in the context of assays for testingcompounds that modulate activity of a PAK2 protein includes thedetermination of a parameter that is indirectly or directly under theinfluence of PAK2, e.g., an indirect, chemical or phenotypic effect suchas inhibition of T lymphocyte activation represented by a change inexpression of a cell surface marker or cytokine production upon TCRstimulation, or changes in cellular proliferation or apoptosis,serine/threonine kinase activity, or TCR signal transduction leading toincreases in intracellular calcium or calcium influx; or, e.g., adirect, physical effect such as ligand binding or inhibition of ligandbinding to PAK2 or a PAK2 domain such as the kinase or crib domain. Afunctional effect therefore includes ligand binding activity, theability of cells to proliferate, apoptosis, gene expression in cellsundergoing activation, serine/threonine kinase activity, expression ofcell surface molecules such as CD69, CD40L and NFAT, TCR signaltransduction, including downstream effectors such as second messengers,intracellular calcium release and calcium influx, production ofcytokines such as IL-2, and other characteristics of activatedlymphocytes. “Functional effects” include in vitro, in vivo, and ex vivoactivities.

By “determining the functional effect” is meant assaying for a compoundthat increases or decreases a parameter that is indirectly or directlyunder the influence of PAK2 protein, e.g., measuring physical andchemical or phenotypic effects. Such functional effects can be measuredby any means known to those skilled in the art, e.g., changes inspectroscopic (e.g., fluorescence, absorbance, refractive index),hydrodynamic (e.g., shape), chromatographic, or solubility propertiesfor the protein; measuring inducible markers or transcriptionalactivation of the protein; measuring binding activity or binding assays,e.g. binding to antibodies; measuring changes in ligand bindingaffinity, e.g., GTPase binding, e.g., Cdc42/Rac or analogs thereof,either naturally occurring or synthetic; measuring cellularproliferation; measuring apoptosis; measuring cell surface markerexpression, e.g., CD69, CD40L and NFAT; measuring cytokine, e.g., IL-2,production; measurement of changes in protein levels for PAK2-associatedsequences; measurement of RNA stability; phosphorylation ordephosphorylation; serine/threonine kinase activity; TCR signaltransduction and downstream effectors, e.g., receptor-ligandinteractions, second messenger concentrations (e.g., cAMP, IP3, orintracellular Ca²⁺); calcium influx; identification of downstream orreporter gene expression (CAT, luciferase, β-gal, GFP and the like),e.g., via chemiluminescence, fluorescence, colorimetric reactions,antibody binding, inducible markers, and ligand binding assays.

“Inhibitors”, “activators”, and “modulators” of PAK2 polynucleotide andpolypeptide sequences are used to refer to activating, inhibitory, ormodulating molecules identified using in vitro and in vivo assays ofPAK2 polynucleotide and polypeptide sequences. Inhibitors are compoundsthat, e.g., bind to, partially or totally block activity, decrease,prevent, delay activation, inactivate, desensitize, or down regulate theactivity or expression of PAK2 proteins, e.g., antagonists. “Activators”are compounds that increase, open, activate, facilitate, enhanceactivation, sensitize, agonize, or up regulate PAK2 protein activity.Inhibitors, activators, or modulators also include genetically modifiedversions of PAK2 proteins, e.g., versions with altered activity, as wellas naturally occurring and synthetic ligands, antagonists, agonists,peptides, cyclic peptides, nucleic acids, antibodies, antisensemolecules, ribozymes, small organic molecules and the like. Such assaysfor inhibitors and activators include, e.g., expressing PAK2 protein invitro, in cells, cell extracts, or cell membranes, applying putativemodulator compounds, and then determining the functional effects onactivity, as described above.

Samples or assays comprising PAK2 proteins that are treated with apotential activator, inhibitor, or modulator are compared to controlsamples without the inhibitor, activator, or modulator to examine theextent of inhibition. Control samples (untreated with inhibitors) areassigned a relative protein activity value of 100%. Inhibition of PAK2is achieved when the activity value relative to the control is about80%, preferably 50%, more preferably 25-0%. Activation of PAK2 isachieved when the activity value relative to the control (untreated withactivators) is 110%, more preferably 150%, more preferably 200-500%(i.e., two to five fold higher relative to the control), more preferably1000-3000% higher.

The term “test compound” or “drug candidate” or “modulator” orgrammatical equivalents as used herein describes any molecule, eithernaturally occurring or synthetic, e.g., protein, oligopeptide (e.g.,from about 5 to about 25 amino acids in length, preferably from about 10to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 aminoacids in length), small organic molecule, polysaccharide, lipid (e.g., asphingolipid), fatty acid, polynucleotide, oligonucleotide, etc., to betested for the capacity to directly or indirectly modulation lymphocyteactivation. The test compound can be in the form of a library of testcompounds, such as a combinatorial or randomized library that provides asufficient range of diversity. Test compounds are optionally linked to afusion partner, e.g., targeting compounds, rescue compounds,dimerization compounds, stabilizing compounds, addressable compounds,and other functional moieties. Conventionally, new chemical entitieswith useful properties are generated by identifying a test compound(called a “lead compound”) with some desirable property or activity,e.g., inhibiting activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds. Often,high throughput screening (HTS) methods are employed for such ananalysis.

A “small organic molecule” refers to an organic molecule, eithernaturally occurring or synthetic, that has a molecular weight of morethan about 50 daltons and less than about 2500 daltons, preferably lessthan about 2000 daltons, preferably between about 100 to about 1000daltons, more preferably between about 200 to about 500 daltons.

“Biological sample” include sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include blood, sputum, tissue, cultured cells, e.g., primarycultures, explants, and transformed cells, stool, urine, etc. Abiological sample is typically obtained from a eukaryotic organism, mostpreferably a mammal such as a primate e.g., chimpanzee or human; cow;dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird;reptile; or fish.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region (e.g., a nucleotide sequence of SEQ ID NO:1), whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web sitehttp://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are thensaid to be “substantially identical.” This definition also refers to, ormay be applied to, the compliment of a test sequence. The definitionalso includes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nu

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I. The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains, e.g., transmembranedomains, pore domains, and cytoplasmic tail domains. Domains areportions of a polypeptide that form a compact unit of the polypeptideand are typically 15 to 350 amino acids long. Exemplary domains includeextracellular domains, transmembrane domains, and cytoplasmic domains.Typical domains are made up of sections of lesser organization such asstretches of β-sheet and α-helices. “Tertiary structure” refers to thecomplete three dimensional structure of a polypeptide monomer.“Quaternary structure” refers to the three dimensional structure formedby the noncovalent association of independent tertiary units.Anisotropic terms are also known as energy terms.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins whichcan be made detectable, e.g., by incorporating a radiolabel into thepeptide or used to detect antibodies specifically reactive with thepeptide.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous reference, e.g., andCurrent Protocols in Molecular Biology, ed. Ausubel, et al.

For PCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y.).

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains; each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). The genes encoding the heavy and lightchains of an antibody of interest can be cloned from a cell, e.g., thegenes encoding a monoclonal antibody can be cloned from a hybridoma andused to produce a recombinant monoclonal antibody. Gene librariesencoding heavy and light chains of monoclonal antibodies can also bemade from hybridoma or plasma cells. Random combinations of the heavyand light chain gene products generate a large pool of antibodies withdifferent antigenic specificity (see, e.g., Kuby, Immunology (3^(rd) ed.1997)). Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No.4,816,567) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized or human antibodies (see,e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992);Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996);Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage displaytechnology can be used to identify antibodies and heteromeric Fabfragments that specifically bind to selected antigens (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as import residues,which are typically taken from an import variable domain. Humanizationcan be essentially performed following the method of Winter andco-workers (see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596(1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such humanizedantibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

In one embodiment, the antibody is conjugated to an “effector” moiety.The effector moiety can be any number of molecules, including labelingmoieties such as radioactive labels or fluorescent labels, or can be atherapeutic moiety. In one aspect the antibody modulates the activity ofthe protein.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, often in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein atleast two times the background and more typically more than 10 to 100times background. Specific binding to an antibody under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. For example, polyclonal antibodies raised to PAK2protein as encoded by SEQ ID NO:1-4, polymorphic variants, alleles,orthologs, and conservatively modified variants, or splice variants, orportions thereof, can be selected to obtain only those polyclonalantibodies that are specifically immunoreactive with PAK2 proteins andnot with other proteins. This selection may be achieved by subtractingout antibodies that cross-react with other molecules. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select antibodies specificallyimmunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, ALaboratory Manual (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity).

By “therapeutically effective dose” herein is meant a dose that produceseffects for which it is administered. The exact dose will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations(1999)).

Assays for Proteins that Modulation T Lymphocyte Activation

High throughput functional genomics assays can be used to identifymodulators of T lymphocyte activation. Such assays can monitor changesin cell surface marker expression, cytokine production, antibodyproduction, proliferation and differentiation, and apoptosis, usingeither cell lines or primary cells. Typically, the lymphocytes arecontacted with a cDNA, a random peptide library (encoded by nucleicacids), or a cyclic peptide library (see, e.g., U.S. Pat. No.6,153,380). The cDNA library can comprise sense, antisense, full length,and truncated cDNAs. The peptide library (optionally cyclic peptides) isencoded by nucleic acids. The lymphocytes are then activated, e.g., byactivating the T cell receptor (TCR, also known as CD3), e.g., usingantibodies to the receptor. The effect of the cDNA or peptide library onthe phenotype of lymphocyte activation is then monitored, using an assayas described above. The effect of the cDNA or peptide can be validatedand distinguished from somatic mutations, using, e.g., regulatableexpression of the nucleic acid such as expression from a tetracyclinepromoter. cDNAs and nucleic acids encoding peptides can be rescued usingtechniques known to those of skill in the art, e.g., using a sequencetag.

Proteins interacting with the peptide or with the protein encoded by thecDNA (e.g., PAK2) can be isolated using a yeast two-hybrid system,mammalian two hybrid system, or phage display screen, etc. Targets soidentified can be further used as bait in these assays to identifyadditional members of the lymphocyte activation pathway, which membersare also targets for drug development (see, e.g., Fields et al., Nature340:245 (1989); Vasavada et al., Proc. Nat'l Acad. Sci. USA 88:10686(1991); Fearon et al., Proc. Nat'l Acad. Sci. USA 89:7958 (1992); Danget al., Mol. Cell. Biol. 11:954 (1991); Chien et al., Proc. Nat'l Acad.Sci. USA 9578 (1991); and U.S. Pat. Nos. 5,283,173, 5,667,973,5,468,614, 5,525,490, and 5,637,463).

Suitable T cell lines include Jurkat, HPB-ALL, HSB-2, and PEER, as wellas other mature and immature T cell lines and primary T cells known tothose of skill in the art. Suitable T cell surface markers include MHCclass II, CD2, CD3, CD4, CD5, CD8, CD25, CD28, CD69, CD40L, LFA-1, andICAM-1 as well as other cell surface markers known to those of skill inthe art (see, e.g., Yablonski et al., Science 281:413-416 (1998)).Suitable cytokines, for measuring either production or response, includeIL-2, IL-4, IL-5, IL-6, IL-10, INF-γ, and TGF-β, as well as theircorresponding receptors.

Cell surface markers can be assayed using fluorescently labeledantibodies and FACS. Cell proliferation can be measured using³H-thymidine or dye inclusion. Apoptosis can be measured using dyeinclusion, or by assaying for DNA laddering or increases inintracellular calcium. Cytokine production can be measured using animmunoassay such as ELISA.

cDNA libraries are made from any suitable source, preferably fromprimary human lymphoid organs such as thymus, spleen, lymph node, andbone marrow. Libraries encoding random peptides are made according totechniques well known to those of skill in the art (see, e.g., U.S. Pat.Nos. 6,153,380, 6,114,111, and 6,180,343). Any suitable vector can beused for the cDNA and peptide libraries, including, e.g., retroviralvectors.

In a preferred embodiment, target proteins that modulate T cellactivation are identified using a high throughput cell based assay(using a microtiter plate format) and FACS screening for CD69 cellsurface expression (see Example I). cDNA libraries are made from primarylymphocyte organs. These cDNA libraries include, e.g., sense, antisense,full length, and truncated cDNAs. The cDNAs are cloned into a retroviralvector with a tet-regulatable promoter. Jurkat cells are infected withthe library, the cells are stimulated with anti-TCR antibodies, and thenthe cells are sorted using fluorescent antibodies and FACS for CD69low/CD3+ cells. Cells with the desired phenotype are recovered,expanded, and cloned. A Tet-regulatable phenotype is established todistinguish somatic mutations. The cDNA is rescued. Optionally, thephenotype is validated by assaying for IL-2 production using primarylymphocytes. Optionally, a marker such as GFP can be used to select forretrovirally infected cells. Using this system, cDNAs encoding PAK2 wereidentified as inhibitors of T cell activation.

Isolation of Nucleic Acids Encoding PAK2 Family Members

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

PAK2 nucleic acids, polymorphic variants, orthologs, and alleles thatare substantially identical to an amino acid sequence encoded by SEQ IDNO:1, as well as other PAK2 family members, can be isolated using PAK2nucleic acid probes and oligonucleotides under stringent hybridizationconditions, by screening libraries. Alternatively, expression librariescan be used to clone PAK2 protein, polymorphic variants, orthologs, andalleles by detecting expressed homologs immunologically with antisera orpurified antibodies made against human PAK2 or portions thereof.

To make a cDNA library, one should choose a source that is rich in PAK2RNA. The mRNA is then made into cDNA using reverse transcriptase,ligated into a recombinant vector, and transfected into a recombinanthost for propagation, screening and cloning. Methods for making andscreening cDNA libraries are well known (see, e.g., Gubler & Hoffman,Gene 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl.Acad. Sci. USA., 72:3961-3965 (1975).

An alternative method of isolating PAK2 nucleic acid and its orthologs,alleles, mutants, polymorphic variants, and conservatively modifiedvariants combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Inniset al., eds, 1990)). Methods such as polymerase chain reaction (PCR) andligase chain reaction (LCR) can be used to amplify nucleic acidsequences of human PAK2 directly from mRNA, from cDNA, from genomiclibraries or cDNA libraries. Degenerate oligonucleotides can be designedto amplify PAK2 homologs using the sequences provided herein.Restriction endonuclease sites can be incorporated into the primers.Polymerase chain reaction or other in vitro amplification methods mayalso be useful, for example, to clone nucleic acid sequences that codefor proteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of PAK2 encoding mRNA in physiological samples,for nucleic acid sequencing, or for other purposes. Genes amplified bythe PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of PAK2 can also be analyzed by techniques known in theart, e.g., reverse transcription and amplification of mRNA, isolation oftotal RNA or poly A⁺ RNA, northern blotting, dot blotting, in situhybridization, RNase protection, high density polynucleotide arraytechnology, e.g., and the like.

Nucleic acids encoding PAK2 protein can be used with high densityoligonucleotide array technology (e.g., GeneChip™) to identify PAK2protein, orthologs, alleles, conservatively modified variants, andpolymorphic variants in this invention. In the case where the homologsbeing identified are linked to modulation of T cell activation, they canbe used with GeneChip™ as a diagnostic tool in detecting the disease ina biological sample, see, e.g., Gunthand et al., AIDS Res. Hum.Retroviruses 14: 869-876 (1998); Kozal et al., Nat. Med. 2:753-759(1996); Matson et al., Anal. Biochem. 224:110-106 (1995); Lockhart etal., Nat. Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res.8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866 (1998).

The gene for PAK2 is typically cloned into intermediate vectors beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. These intermediate vectors are typically prokaryotevectors, e.g., plasmids, or shuttle vectors.

Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding PAK2, one typically subclones PAK2 into an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al., and Ausubel et al, supra. Bacterial expressionsystems for expressing the PAK2 protein are available in, e.g., E. coli,Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available. In one preferred embodiment, retroviralexpression systems are used in the present invention.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the PAK2 encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encoding PAK2and signals required for efficient polyadenylation of the transcript,ribosome binding sites, and translation termination. Additional elementsof the cassette may include enhancers and, if genomic DNA is used as thestructural gene, introns with functional splice donor and acceptorsites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as MBP, GST, and LacZ. Epitope tags can also beadded to recombinant proteins to provide convenient methods ofisolation, e.g., c-myc. Sequence tags may be included in an expressioncassette for nucleic acid rescue. Markers such as fluorescent proteins,green or red fluorescent protein, β-gal, CAT, and the like can beincluded in the vectors as markers for vector transduction.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, retroviral vectors, and vectorsderived from Epstein-Barr virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe CMV promoter, SV40 early promoter, SV40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Expression of proteins from eukaryotic vectors can be also be regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal.

In one embodiment, the vectors of the invention have a regulatablepromoter, e.g., tet-regulated systems and the RU-486 system (see, e.g.,Gossen & Bujard, Proc. Nat'l Acad. Sci. USA 89:5547 (1992); Oligino etal., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441(1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al.,Nat. Biotechnol. 16:757-761 (1998)). These impart small molecule controlon the expression of the candidate target nucleic acids. This beneficialfeature can be used to determine that a desired phenotype is caused by atransfected cDNA rather than a somatic mutation.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with aPAK2 encoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of PAK2protein, which are then purified using standard techniques (see, e.g.,Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,biolistics, liposomes, microinjection, plasma vectors, viral vectors andany of the other well known methods for introducing cloned genomic DNA,cDNA, synthetic DNA or other foreign genetic material into a host cell(see, e.g., Sambrook et al., supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingPAK2.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofPAK2, which is recovered from the culture using standard techniquesidentified below.

Purification of PAK2 Polypeptides

Either naturally occurring or recombinant PAK2 can be purified for usein functional assays. Naturally occurring PAK2 can be purified, e.g.,from human tissue. Recombinant PAK2 can be purified from any suitableexpression system.

The PAK2 protein may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification: Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant PAK2 protein isbeing purified. For example, proteins having established molecularadhesion properties can be reversible fused to the PAK2 protein. Withthe appropriate ligand, PAK2 protein can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. Finally,PAK2 protein could be purified using immunoaffinity columns.

A. Purification of PAK2 from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is one example of aninducible promoter system. Bacteria are grown according to standardprocedures in the art. Fresh or frozen bacteria cells are used forisolation of protein.

Proteins expressed in bacteria may form insoluble aggregates (“inclusionbodies”). Several protocols are suitable for purification of PAK2protein inclusion bodies. For example, purification of inclusion bodiestypically involves the extraction, separation and/or purification ofinclusion bodies by disruption of bacterial cells, e.g., by incubationin a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgCl₂, 1 mM DTT,0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3passages through a French Press, homogenized using a Polytron (BrinkmanInstruments) or sonicated on ice. Alternate methods of lysing bacteriaare apparent to those of skill in the art (see, e.g., Sambrook et al.,supra; Ausubel et al., supra).

If necessary, the inclusion bodies are solubilized, and the lysed cellsuspension is typically centrifuged to remove unwanted insoluble matter.Proteins that formed the inclusion bodies may be renatured by dilutionor dialysis with a compatible buffer. Suitable solvents include, but arenot limited to urea (from about 4 M to about 8 M), formamide (at leastabout 80%, volume/volume basis), and guanidine hydrochloride (from about4 M to about 8 M). Some solvents which are capable of solubilizingaggregate-forming proteins, for example SDS (sodium dodecyl sulfate),70% formic acid, are inappropriate for use in this procedure due to thepossibility of irreversible denaturation of the proteins, accompanied bya lack of immunogenicity and/or activity. Although guanidinehydrochloride and similar agents are denaturants, this denaturation isnot irreversible and renaturation may occur upon removal (by dialysis,for example) or dilution of the denaturant, allowing reformation ofimmunologically and/or biologically active protein. Other suitablebuffers are known to those skilled in the art. Human PAK2 proteins areseparated from other bacterial proteins by standard separationtechniques, e.g., with Ni-NTA agarose resin.

Alternatively, it is possible to purify PAK2 protein from bacteriaperiplasm. After lysis of the bacteria, when the PAK2 protein exportedinto the periplasm of the bacteria, the periplasmic fraction of thebacteria can be isolated by cold osmotic shock in addition to othermethods known to skill in the art. To isolate recombinant proteins fromthe periplasm, the bacterial cells are centrifuged to form a pellet. Thepellet is resuspended in a buffer containing 20% sucrose. To lyse thecells, the bacteria are centrifuged and the pellet is resuspended inice-cold 5 mM MgSO₄ and kept in an ice bath for approximately 10minutes. The cell suspension is centrifuged and the supernatant decantedand saved. The recombinant proteins present in the supernatant can beseparated from the host proteins by standard separation techniques wellknown to those of skill in the art.

B. Standard Protein Separation Techniques for Purifying PAK2 Proteins.

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of the PAK2 proteins can be used to isolate it fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

The PAK2 proteins can also be separated from other proteins on the basisof its size, net surface charge, hydrophobicity, and affinity forligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art. It will be apparent to one ofskill that chromatographic techniques can be performed at any scale andusing equipment from many different manufacturers (e.g., PharmaciaBiotech).

Assays for Modulators of PAK2 Protein

A. Assays

Modulation of a PAK2 protein, and corresponding modulation of Tlymphocyte activation and TCR signaling, can be assessed using a varietyof in vitro and in vivo assays, including cell-based models as describedabove. Such assays can be used to test for inhibitors and activators ofPAK2 protein, and, consequently, inhibitors and activators of lymphocyteactivation. Such modulators of PAK2 protein, which is involved in Tlymphocyte activation and TCR signaling, are useful for treatingdisorders related to T cell activation. Modulators of PAK2 protein aretested using either recombinant or naturally occurring PAK2, preferablyhuman PAK2.

Preferably, the PAK2 protein will have the sequence as encoded by SEQ IDNO:2 or a conservatively modified variant thereof. Alternatively, thePAK2 protein of the assay will be derived from a eukaryote and includean amino acid subsequence having substantial amino acid sequenceidentity to SEQ ID NO:2. Generally, the amino acid sequence identitywill be at least 60%, preferably at least 65%, 70%, 75%, 80%, 85%, or90%, most preferably at least 95%.

Measurement of lymphocyte activation or loss-of-T lymphocyte activationphenotype on PAK2 protein or cell expressing PAK2 protein, eitherrecombinant or naturally occurring, can be performed using a variety ofassays, in vitro, in vivo, and ex vivo, as described herein. A suitablephysical, chemical or phenotypic change that affects activity or bindingcan be used to assess the influence of a test compound on thepolypeptide of this invention. When the functional effects aredetermined using intact cells or animals, one can also measure a varietyof effects such as, in the case of signal transduction, e.g., ligandbinding, hormone release, transcriptional changes to both known anduncharacterized genetic markers (e.g., northern blots), changes in cellmetabolism such as pH changes, serine/threonine kinase activity, andchanges signal transduction such as changes in intracellular secondmessengers such as Ca²⁺, IP3, cGMP, or cAMP; as well as changes relatedto lymphocyte activation, e.g., cellular proliferation, cell surfacemarker expression, e.g., CD69, CD40L and NFAT, cytokine production,e.g., IL-2, and apoptosis.

In one preferred embodiment, described herein in Example I, measurementof CD69 activation and FACS sorting is used to identify modulators of Tcell activation.

In Vitro Assays

Assays to identify compounds with PAK2 modulating activity can beperformed in vitro. Such assays can used full length PAK2 protein or avariant thereof (see, e.g., SEQ ID NOS:1-4), or a fragment of a PAK2protein, such as the kinase or crib domain. Purified recombinant ornaturally occurring PAK2 protein or fragments thereof can be used in thein vitro methods of the invention. In addition to purified PAK2 protein,the recombinant or naturally occurring PAK2 protein can be part of acellular lysate. As described below, the assay can be either solid stateor soluble. Preferably, the protein is bound to a solid support, eithercovalently or non-covalently. Often, the in vitro assays of theinvention are ligand binding or ligand affinity assays, eithernon-competitive or competitive (with known ligands such as Cdc42/Rac).Other in vitro assays include measuring changes in spectroscopic (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties for the protein. In oneembodiment, the in vitro assay measures PAK2 serine/threonine kinaseactivity.

In one embodiment, a high throughput binding assay is performed in whichthe PAK2 protein or a fragment thereof such as a crib or kinase domainis contacted with a potential modulator and incubated for a suitableamount of time. In one embodiment, the potential modulator is bound to asolid support, and the PAK2 protein is added. In another embodiment, thePAK2 protein is bound to a solid support. A wide variety of modulatorscan be used, as described below, including small organic molecules,peptides, antibodies, and PAK2 ligand analogs. A wide variety of assayscan be used to identify PAK2-modulator binding, including labeledprotein-protein binding assays, electrophoretic mobility shifts,immunoassays, enzymatic assays such as phosphorylation assays, and thelike. In some cases, the binding of the candidate modulator isdetermined through the use of competitive binding assays, whereinterference with binding of a known ligand is measured in the presenceof a potential modulator. Ligands for PAK2 family are known (e.g.,Cdc42/Rac). Either the modulator or the known ligand is bound first, andthen the competitor is added. After the PAK2 protein is washed,interference with binding, either of the potential modulator or of theknown ligand, is determined. Often, either the potential modulator orthe known ligand is labeled.

Cell-Based in Vivo Assays

In another embodiment, PAK2 protein is expressed in a cell, andfunctional, e.g., physical and chemical or phenotypic, changes areassayed to identify PAK2 and lymphocyte activation modulators. Cellsexpressing PAK2 proteins can also be used in binding assays. Anysuitable functional effect can be measured, as described herein. Forexample, ligand binding, cell surface marker expression, cellularproliferation, apoptosis, cytokine production, serine/threonine kinaseactivity, and GTPase binding, are all suitable assays to identifypotential modulators using a cell based system. Suitable cells for suchcell based assays include both primary lymphocytes and cell lines, asdescribed herein. The PAK2 protein can be naturally occurring orrecombinant.

As described above, in one embodiment, lymphocyte activation is measuredby contacting T cells comprising a PAK2 target with a potentialmodulator and activating the cells with an anti-TCR antibody. Modulationof T cell activation is identified by screening for cell surface markerexpression, e.g., CD69 expression levels, using fluorescent antibodiesand FACS sorting.

In another embodiment, cellular proliferation or apoptosis can bemeasured using ³H-thymidine incorporation or dye inclusion. Cytokineproduction can be measured using an immunoassay such as an ELISA.

In another embodiment, cellular PAK2 polypeptide levels are determinedby measuring the level of protein or mRNA. The level of PAK2 protein orproteins related to PAK2 signal transduction are measured usingimmunoassays such as western blotting, ELISA and the like with anantibody that selectively binds to the PAK2 polypeptide or a fragmentthereof. For measurement of mRNA, amplification, e.g., using PCR, LCR,or hybridization assays, e.g., northern hybridization, RNAse protection,dot blotting, are preferred. The level of protein or mRNA is detectedusing directly or indirectly labeled detection agents, e.g.,fluorescently or radioactively labeled nucleic acids, radioactively orenzymatically labeled antibodies, and the like, as described herein.

Signal transduction related to TCR signaling can also be measured.Activated or inhibited TCR signaling will alter the properties of targetenzymes, second messengers, channels, and other effector proteins. Theexamples include the activation of cGMP phosphodiesterase, adenylatecyclase, phospholipase C, IP3, and modulation of diverse channels.Downstream consequences can also be examined such as generation ofdiacyl glycerol and IP3 by phospholipase C, and in turn, for calciummobilization by IP3. For example, changes in Ca²⁺ levels are optionallymeasured using fluorescent Ca²⁺ indicator dyes and fluorometric imaging.

Alternatively, PAK2 expression can be measured using a reporter genesystem. Such a system can be devised using a PAK2 protein promoteroperably linked to a reporter gene such as chloramphenicolacetyltransferase, firefly luciferase, bacterial luciferase,β-galactosidase and alkaline phosphatase. Furthermore, the protein ofinterest can be used as an indirect reporter via attachment to a secondreporter such as red or green fluorescent protein (see, e.g., Mistili &Spector, Nature Biotechnology 15:961-964 (1997)). The reporter constructis typically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art.

Animal Models

Animal models of lymphocyte activation also find use in screening formodulators of lymphocyte activation. Similarly, transgenic animaltechnology including gene knockout technology, for example as a resultof homologous recombination with an appropriate gene targeting vector,or gene overexpression, will result in the absence or increasedexpression of the PAK2 protein. When desired, tissue-specific expressionor knockout of the PAK2 protein may be necessary. Transgenic animalsgenerated by such methods find use as animal models of lymphocyteactivation and are additionally useful in screening for modulators oflymphocyte activation.

Knock-out cells and transgenic mice can be made by insertion of a markergene or other heterologous gene into the endogenous PAK2 gene site inthe mouse genome via homologous recombination. Such mice can also bemade by substituting the endogenous PAK2 with a mutated version of PAK2,or by mutating the endogenous PAK2, e.g., by exposure to carcinogens.

A DNA construct is introduced into the nuclei of embryonic stem cells.Cells containing the newly engineered genetic lesion are injected into ahost mouse embryo, which is re-implanted into a recipient female. Someof these embryos develop into chimeric mice that possess germ cellspartially derived from the mutant cell line. Therefore, by breeding thechimeric mice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric targeted mice can be derived according to Hogan etal., Manipulating the Mouse Embryo: A Laboratory Manual, Cold SpringHarbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, Robertson, ed., IRL Press, Washington, D.C.,(1987).

B. Modulators

The compounds tested as modulators of PAK2 protein can be any smallorganic molecule, or a biological entity, such as a protein, e.g., anantibody or peptide, a sugar, a nucleic acid, e.g., an antisenseoligonucleotide or a ribozyme, or a lipid. Alternatively, modulators canbe genetically altered versions of a PAK2 protein. Typically, testcompounds will be small organic molecules, peptides, lipids, and lipidanalogs. In one embodiment, a modulator is a trans-dominant peptidefragment of the PAK2 kinase domain, which binds to and inactivates thePAK2 kinase domain.

Essentially any chemical compound can be used as a potential modulatoror ligand in the assays of the invention, although most often compoundscan be dissolved in aqueous or organic (especially DMSO-based) solutionsare used. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involveproviding a combinatorial small organic molecule or peptide librarycontaining a large number of potential therapeutic compounds (potentialmodulator or ligand compounds). Such “combinatorial chemical libraries”or “ligand libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries. (see,e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res.37:487-493 (1991) and Houghton et al., Nature 354:84-88 (1991)). Otherchemistries for generating chemical diversity libraries can also beused. Such chemistries include, but are not limited to: peptoids (e.g.,PCT Publication No. WO 91/19735), encoded peptides (e.g., PCTPublication No. WO 93/20242), random bio-oligomers (e.g., PCTPublication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No.5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding(Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogousorganic syntheses of small compound libraries (Chen et al., J. Amer.Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho et al., Science261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org.Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger andSambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S.Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al.,Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287),carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522(1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries(see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

C. Solid State and Soluble High Throughput Assays

In one embodiment the invention provides soluble assays using a PAK2protein or a fragment thereof such as the kinase or crib domain, or acell or tissue expressing a PAK2 protein, either naturally occurring orrecombinant. In another embodiment, the invention provides solid phasebased in vitro assays in a high throughput format, where the PAK2protein or fragment thereof, such as the kinase or crib domain, isattached to a solid phase substrate. Any one of the assays describedherein can be adapted for high throughput screening, e.g., ligandbinding, cellular proliferation, cell surface marker flux, e.g., CD-69screening, kinase activity, second messenger flux, e.g., Ca²⁺, IP3,cGMP, or cAMP, cytokine production, etc. In one preferred embodiment,the cell-based system using CD-69 modulation and FACS assays is used ina high throughput format for identifying modulators of PAK2 proteins,and therefore modulators of T cell activation. In another preferredembodiment, the kinase domain or the crib domain of PAK2 is used in highthroughput in vitro binding assays for modulators.

In the high throughput assays of the invention, either soluble or solidstate, it is possible to screen up to several thousand differentmodulators or ligands in a single day. This methodology can be used forPAK2 proteins in vitro, or for cell-based or membrane-based assayscomprising a PAK2 protein. In particular, each well of a microtiterplate can be used to run a separate assay against a selected potentialmodulator, or, if concentration or incubation time effects are to beobserved, every 5-10 wells can test a single modulator. Thus, a singlestandard microtiter plate can assay about 100 (e.g., 96) modulators. If1536 well plates are used, then a single plate can easily assay fromabout 100-about 1500 different compounds. It is possible to assay manyplates per day; assay screens for up to about 6,000, 20,000, 50,000, ormore than 100,000 different compounds are possible using the integratedsystems of the invention.

For a solid state reaction, the protein of interest or a fragmentthereof, e.g., an extracellular domain, or a cell or membrane comprisingthe protein of interest or a fragment thereof as part of a fusionprotein can be bound to the solid state component, directly orindirectly, via covalent or non covalent linkage e.g., via a tag. Thetag can be any of a variety of components. In general, a molecule whichbinds the tag (a tag binder) is fixed to a solid support, and the taggedmolecule of interest is attached to the solid support by interaction ofthe tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders; see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs. For example, agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherein family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g. which mediate the effects of various smallligands, including steroids, thyroid hormone, retinoids and vitamin D;peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclicpolymer configurations), oligosaccharides, proteins, phospholipids andantibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly gly sequencesof between about 5 and 200 amino acids. Such flexible linkers are knownto persons of skill in the art. For example, poly(ethelyne glycol)linkers are available from Shearwater Polymers, Inc. Huntsville, Ala.These linkers optionally have amide linkages, sulfhydryl linkages, orheterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature. See, e.g., Merrifield, J.Am. Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank &Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal et al., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of L biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

Immunological Detection of PAK2 Polypeptides

In addition to the detection of PAK2 gene and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect PAK2 proteins of the invention. Such assays are useful forscreening for modulators of PAK2 and lymphocyte activation, as well asfor therapeutic and diagnostic applications. Immunoassays can be used toqualitatively or quantitatively analyze PAK2 protein. A general overviewof the applicable technology can be found in Harlow & Lane, Antibodies:A Laboratory Manual (1988).

A. Production of Antibodies

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with the PAK2 proteins are known to those of skill in theart (see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow& Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice(2d ed. 1986); and Kohler & Milstein, Nature 256:495-497 (1975). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors, as wellas preparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of immunogens comprising portions of PAK2 protein may be usedto produce antibodies specifically reactive with PAK2 protein. Forexample, recombinant PAK2 protein or an antigenic fragment thereof, canbe isolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies may be generated,for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see, Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler & Milstein, Eur. J. Immunol. 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse, etal., Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-PAK2proteins, using a competitive binding immunoassay. Specific polyclonalantisera and monoclonal antibodies will usually bind with a K_(d) of atleast about 0.1 mM, more usually at least about 1 μM, preferably atleast about 0.1 μM or better, and most preferably, 0.01 μM or better.Antibodies specific only for a particular PAK family member, such asPAK2, or a particular PAK2 ortholog, such as human PAK2, can also bemade, by subtracting out other cross-reacting PAK family members ororthologs from a species such as a non-human mammal. In this manner,antibodies that bind only to a particular PAK protein or ortholog may beobtained.

Once the specific antibodies against PAK2 protein are available, theprotein can be detected by a variety of immunoassay methods. Inaddition, the antibody can be used therapeutically as a PAK2 modulators.For a review of immunological and immunoassay procedures, see Basic andClinical Immunology (Stites & Terr eds., 7^(th) ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio, ed., 1980); and Harlow & Lane, supra.

B. Immunological Binding Assays

PAK2 protein can be detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology: Antibodies inCell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology(Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (orimmunoassays) typically use an antibody that specifically binds to aprotein or antigen of choice (in this case the PAK2 protein or antigenicsubsequence thereof). The antibody (e.g., anti-PAK2) may be produced byany of a number of means well known to those of skill in the art and asdescribed above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled PAK2 or a labeledanti-PAK2 antibody. Alternatively, the labeling agent may be a thirdmoiety, such a secondary antibody, that specifically binds to theantibody/PAK2 complex (a secondary antibody is typically specific toantibodies of the species from which the first antibody is derived).Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G may also be used as the labelagent. These proteins exhibit a strong non-immunogenic reactivity withimmunoglobulin constant regions from a variety of species (see, e.g.,Kronval et al., J. Immunol. 111: 1401-1406 (1973); Akerstrom et al., J.Immunol. 135:2589-2542 (1985)). The labeling agent can be modified witha detectable moiety, such as biotin, to which another molecule canspecifically bind, such as streptavidin. A variety of detectablemoieties are well known to those skilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, optionally from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting PAK2 in samples may be either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In one preferred “sandwich”assay, for example, the anti-PAK2 antibodies can be bound directly to asolid substrate on which they are immobilized. These immobilizedantibodies then capture PAK2 present in the test sample. PAK2 proteinsthus immobilized are then bound by a labeling agent, such as a secondPAK2 antibody bearing a label. Alternatively, the second antibody maylack a label, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second or third antibody is typically modified with adetectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive Assay Formats

In competitive assays, the amount of PAK2 protein present in the sampleis measured indirectly by measuring the amount of a known, added(exogenous) PAK2 protein displaced (competed away) from an anti-PAK2antibody by the unknown PAK2 protein present in a sample. In onecompetitive assay, a known amount of PAK2 protein is added to a sampleand the sample is then contacted with an antibody that specificallybinds to PAK2 protein. The amount of exogenous PAK2 protein bound to theantibody is inversely proportional to the concentration of PAK2 proteinpresent in the sample. In a particularly preferred embodiment, theantibody is immobilized on a solid substrate. The amount of PAK2 proteinbound to the antibody may be determined either by measuring the amountof PAK2 present in PAK2 protein/antibody complex, or alternatively bymeasuring the amount of remaining uncomplexed protein. The amount ofPAK2 protein may be detected by providing a labeled PAK2 molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known PAK2 protein is immobilized on a solid substrate. Aknown amount of anti-PAK2 antibody is added to the sample, and thesample is then contacted with the immobilized PAK2. The amount ofanti-PAK2 antibody bound to the known immobilized PAK2 is inverselyproportional to the amount of PAK2 protein present in the sample. Again,the amount of immobilized antibody may be detected by detecting eitherthe immobilized fraction of antibody or the fraction of the antibodythat remains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-Reactivity Determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a PAK2 protein can beimmobilized to a solid support. Proteins (e.g., PAK2 and homologs) areadded to the assay that compete for binding of the antisera to theimmobilized antigen. The ability of the added proteins to compete forbinding of the antisera to the immobilized protein is compared to theability of the PAK2 protein to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps an allele or polymorphic variant of a PAK2protein, to the immunogen protein. In order to make this comparison, thetwo proteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required to inhibit 50% of binding is less than 10 timesthe amount of the PAK2 protein that is required to inhibit 50% ofbinding, then the second protein is said to specifically bind to thepolyclonal antibodies generated to PAK2 immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of PAK2 in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind PAK2. The anti-PAK2 antibodies specifically bindto the PAK2 on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the anti-PAK2 antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-Specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize PAK2 protein, orsecondary antibodies that recognize anti-PAK2.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see U.S. Pat. No.4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

Cellular Transfection and Gene Therapy

The present invention provides the nucleic acids of PAK2 protein for thetransfection of cells in vitro and in vivo. These nucleic acids can beinserted into any of a number of well-known vectors for the transfectionof target cells and organisms as described below. The nucleic acids aretransfected into cells, ex vivo or in vivo, through the interaction ofthe vector and the target cell. The nucleic acid, under the control of apromoter, then expresses a PAK2 protein of the present invention,thereby mitigating the effects of absent, partial inactivation, orabnormal expression of a PAK2 gene, particularly as it relates to T cellactivation. The compositions are administered to a patient in an amountsufficient to elicit a therapeutic response in the patient. An amountadequate to accomplish this is defined as “therapeutically effectivedose or amount.”

Such gene therapy procedures have been used to correct acquired andinherited genetic defects, cancer, and other diseases in a number ofcontexts. The ability to express artificial genes in humans facilitatesthe prevention and/or cure of many important human diseases, includingmany diseases which are not amenable to treatment by other therapies(for a review of gene therapy procedures, see Anderson, Science256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani &Caskey, TIBTECH 11:162-166 (1993); Mulligan, Science 926-932 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology (Doerfler & Böhm eds., 1995); and Yu etal., Gene Therapy 1:13-26 (1994)).

Pharmaceutical Compositions and Administration

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered (e.g., nucleic acid, protein,modulatory compounds or transduced cell), as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington's Pharmaceutical Sciences,17^(th) ed., 1989). Administration can be in any convenient manner,e.g., by injection, oral administration, inhalation, transdermalapplication, or rectal administration.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the packaged nucleic acidsuspended in diluents, such as water, saline or PEG 400; (b) capsules,sachets or tablets, each containing a predetermined amount of the activeingredient, as liquids, solids, granules or gelatin; (c) suspensions inan appropriate liquid; and (d) suitable emulsions. Tablet forms caninclude one or more of lactose, sucrose, mannitol, sorbitol, calciumphosphates, corn starch, potato starch, microcrystalline cellulose,gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearicacid, and other excipients, colorants, fillers, binders, diluents,buffering agents, moistening agents, preservatives, flavoring agents,dyes, disintegrating agents, and pharmaceutically compatible carriers.Lozenge forms can comprise the active ingredient in a flavor, e.g.,sucrose, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin or sucrose and acaciaemulsions, gels, and the like containing, in addition to the activeingredient, carriers known in the art.

The compound of choice, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions can be administered, forexample, by intravenous infusion, orally, topically, intraperitoneally,intravesically or intrathecally. Parenteral administration andintravenous administration are the preferred methods of administration.The formulations of commends can be presented in unit-dose or multi-dosesealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Cellstransduced by nucleic acids for ex vivo therapy can also be administeredintravenously or parenterally as described above.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial therapeuticresponse in the patient over time. The dose will be determined by theefficacy of the particular vector employed and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose also will be determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, or transduced cell type in aparticular patient.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant expression of the PAK2 protein, the physician evaluatescirculating plasma levels of the vector, vector toxicities, progressionof the disease, and the production of anti-vector antibodies. Ingeneral, the dose equivalent of a naked nucleic acid from a vector isfrom about 1 μg to 100 μg for a typical 70 kilogram patient, and dosesof vectors which include a retroviral particle are calculated to yieldan equivalent amount of therapeutic nucleic acid.

For administration, compounds and transduced cells of the presentinvention can be administered at a rate determined by the LD-50 of theinhibitor, vector, or transduced cell type, and the side-effects of theinhibitor, vector or cell type at various concentrations, as applied tothe mass and overall health of the patient. Administration can beaccomplished via single or divided doses.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Identification of Genes Involved in Modulation of T CellActivation

A. Introduction

In this study, an approach to identify new targets for immunesuppressive drugs is provided. It is known that following T cellactivation, expression of numerous cell surface markers such as CD25,CD69, and CD40L are upregulated. CD69 has been shown to be an earlyactivation marker in T, B, and NK cells. CD69 is a disulfide-linkeddimer. It is not expressed in resting lymphocytes but appears on T, Band NK cells after activation in vitro. Its relevance as a TCR signalingoutcome has been validated using T cell deficient in certain keysignaling molecules such as LAT and SLP76 (Yablonski, supra).Furthermore, re-introducing SLP76 to the deficient cells results inrestoration of CD69 expression. CD69 upregulation was therefore to beused to monitor TCR signal transduction. The rationale of the functionalgenomics screen was then to identify cell clones whose CD69 upregulationwas repressed following introduction of a retroviral cDNA library. Thelibrary members conferring such repression would then represent immunemodulators that function to block TCR signal transduction.

B. Results

Several T cell lines, including Jurkat, HPB-ALL, HSB-2 and PEER weretested for the presence of surface CD3, CD25, CD28, CD40L, CD69, CD95,and CD95L. Those that express CD3 were cultured with anti-CD3 oranti-TCR to crosslink the TCR and examined for the upregulation of CD69.Jurkat T cell line was selected for its ability to upregulate CD69 inresponse to crosslinking of their TCR with a kinetics mimicking that ofprimary T lymphocytes (data not shown). The population of Jurkat cellswas sorted for low basal and highly inducible CD69 expression followinganti-TCR stimulation. Clone 4D9 was selected because CD69 in this clonewas uniformly and strongly induced following TCR stimulation in 24hours.

In order to regulate the expression of the retroviral library, theTet-Off system was used. Basically, cDNA inserts in the retrovirallibrary were cloned behind the tetracycline regulatory element (TRE) andthe minimal promoter of TK. Transcription of the cDNA inserts were thendependent on the presence of tetracycline-controlled trans-activator(tTA), a fusion of Tet repression protein and the VP16 activationdomain, and the absence of tetracyaline or its derivatives such asdoxycycline (Dox). To shut off the cDNA expression, one can simply adddoxycycline in the medium. To obtain a Jurkat clone stably expressestTA, retroviral LTR-driven tTA was introduced in conjunction with aTRE-dependent reporter construct, namely TRA-Lyt2. Through sorting ofLyt2 positive cells in the absence of Dox and Lyt2 negative cells in thepresence of Dox, coupled with clonal evaluation, a derivative of Jurkatclone 4D9 was obtained, called 4D9#32, that showed the best Doxregulation of Lyt2 expression.

Positive controls: ZAP70 is a positive regulator of T cell activation. Akinase-inactivated (KI) ZAP70 and a truncated ZAP70 (SH2 N+C) weresubcloned into the retroviral vector under TRE control. ZAP70 SH2 (N+C)and ZAP70 KI both inhibited TCR-induced CD69 expression. Consistent withthe published report on dominant negative forms of ZAP70 on NFATactivity, the truncated protein is also a more potent inhibitor of CD69induction. In addition, the higher protein expression, as shown byadjusting GFP-gating, the stronger the inhibition was. When one puts themarker M1 at bottom 1% of the uninfected cells, one has a 40% likelihoodof obtaining cells whose phenotype resembled that of ZAP70 SH2 (N+C).This translates into a 40:1 enrichment of the desired phenotype.

The CD69 inhibitory phenotype is dependent on expression of dominantnegative forms of ZAP70. When Dox was added for 7 days before TCR wasstimulated, there was no inhibition of CD69 expression. Analysis ofcellular phenotype by FACS of GFP, which was produced from thebi-cistronic mRNA ZAP70 SH2 (N+C)-IRES-GFP, revealed a lack of GFP+cells. The lack of ZAP70 SH2 (N+C) expression in the presence of Dox wasconfirmed by Western.

Screening for cells lacking CD69 upregulation: Jurkat 4D9#32 cells wereinfected with cDNA libraries made form primary human lymphoid organssuch as thymus, spleen, lymph node and bone marrow. The librarycomplexity was 5×10⁷ and was built on the TRE vector. A total of 7.1×10⁸cells were screened with an infection rate of 52%, as judged by parallelinfection of the same cells with TRA-dsGFP (data not shown). Afterinfection, the cells will be stimulated with the anti-TCR antibody C305for overnight and sorted for CD69 low and CD3+ phenotype by FACS. If thesorting gate was set to include the bottom 3% cells based on the singleparameter of CD69 level, 2/3 cells in the sorting gate lacked TCR/CD3complex, which explained their refractory to stimulation. The secondparameter of CD3 expression was then incorporated. Even though there wasa significant reduction of CD3/TCR complex on the surface followingreceptor-mediated internalization, the CD3− population was stilldistinguishable from the CD3+ population. The resulting sort gatecontained 1% of the total cells, which translated into a 100-foldenrichment based on cell numbers. The recovered cells with CD69 low CD3+phenotype were allowed to rest in complete medium for 5 days beforebeing stimulated again for a new round of sorting. In subsequent roundof sortings, the sort gate was always maintained to contain theequivalent of 1% of the unsorted control population. Obvious enrichmentwas achieved after 3 rounds of reiterative sorting. Cells with thedesired phenotype increased from 1% to 22.3%. In addition, the overallpopulation's geometric mean for CD69 was also reduced.

In order to ascertain that the phenotype was due to expression of thecDNA library rather than entirely due to spontaneous or retroviralinsertion-mediated somatic mutation, the cells recovered after the thirdround of sorting were split into two halves. One half of the cells weregrown in the absence of Dox while the other half in the presence of Dox.A week later, CD69 expression was compared following anti-TCRstimulation. There was a significant numbers of cells (11%) whose CD69repression was lost in the presence of Dox, suggesting that the CD69inhibition phenotype was indeed caused by the expression of librarymembers. Single cell clones in conjunction with the fourth round of CD69low CD3+ sorting (LLLL) were deposited.

In order to reduce the number of cells whose phenotype was notDox-regulatable, the half of the cells grown in the presence of Dox weresubjected to a fourth round of sorting for enrichment of CD69 highphenotype (LLLH). The cells recovered from LLLH sort were cultured inthe absence of Dox for subsequence sorting and single cell cloning ofCD69 low CD3+ phenotypes.

Dox regulation of CD69 expression was expressed as the ratio ofgeometric mean fluorescent intensity (GMFI) in the presence of Dox overthat in the absence of Dox. In uninfected cells, Dox had limited effecton the induction of CD69 expression so that the ratio of GMFI(+Dox)/GMFI (−Dox) remained to be 1.00+/−0.25. The 2× standard deviationwas therefore used as a cut-off criterion and clones with a ratio above1.5 were regarded as Dox-regulated clones.

RNA samples were prepared from clones with Dox-regulatable phenotypes.Using primers specific for the vector sequence flanking the cDNA libraryinsert, the cDNA insert of selected clones were captured by RT-PCR. Mostclones generated only on DNA band, whereas a few clones generated two ormore bands. Sequencing analysis revealed that the additional bands werecaused by double or multiple insertions.

Characterization of proteins involved in T cell activation: Known TCRregulators such as Lck, ZAP70, PLCγ1 and Raf were obtained. In addition,the BCR regulator SYK was also uncovered. Molecules previously notassociated with TCR activation, such as PAK2, were also identified usingthis screen.

Lck is a non-receptor protein tyrosine kinase. Its role in T celldevelopment and activation has been widely documented. So far, dominantnegative form of Lck has no been reported. Our discovery that overexpression of the kinase-truncated form of Lck caused inhibition ofCD69, similar to the phenotype of Jurkat somatic mutant lacking Lck,suggests that kinase deletion of Lck could also work as a dominantnegative form of Lck.

The two ZAP70 hits ended at aa 262 and 269, respectively. They bothmissed the catalytic domain. The deletions are very close to thepositive control for the screen, ZAP70 SH2 (N+C), which ended at aa 276.Since ZAP70 SH2 (N+C) was shown to be a dominant negative protein, itappears that the two ZAP70 hits also behaved as dominant negativeproteins of ZAP70.

SYK is a non-receptor tyrosine kinase belonging to the SYK/ZAP70 familyof kinases. Since it has also been shown that the lack of SYK expressionin Jurkat cells did not appear to significantly alter the TCR-mediatedresponses compared with Jurkat clones expressing SYK, it appears thatthe SYK hit obtained from our screen worked mainly to block ZAP70function. SYK's similarity to ZAP70 and its ability to associate withphosphorylated TCR zeta chains also support this notion.

PLCγ1 plays a crucial role in coupling T cell receptor ligation to IL-2gene expression in activated T lymphocytes. TCR engagement leads torapid tyrosine phosphorylation and activation of PLCγ1. The activatedenzyme converts phosphatidylinositol-4,5-bisphosphate (PIP2) toinositol-1,3,5-trisphosphate ((IP3) and diacylglycerol (DAG). IP3triggers intracellular Ca2+ increase and DAG is a potent activator ofprotein kinase C (PKC). PLCγ1 has a split catalytic domain comprised ofconserved X and Y subdomains. Single point mutation in the catalytic Xbox completely abolished the enzyme activity and also blocked IL-2reporter gene expression when introduced into PLCγ-deficient Jurkatcells. Our hit contained the PH domain and the N and C terminal SH2domains of PLCγ1. Significantly this hit also deleted the crucialtyrosine Y783 between the SH2 and SH3 domains. It was reported that Y783was essential for coupling of TCR stimulation to IL-2 promoteractivation and that mutation of Y783 to F (phenylalanine) generated avery potent dominant negative form of PLCγ1. Indeed, the original cloneencoding the PLCγ1hit had the highest Dox +/− ratio for CD69 expressionamong all clones from the cDNA screen, indicating the strong repressionof CD69 induction by the hit as well as the total de-repression in theabsence of the hit. When introduced to naïve Jurkat cells, this fragmentcaused severe block of TCR-induced CD69 expression.

Raf is a MAP kinase kinase kinase. It interacts with Ras and leads toactivation of the MAP kinase pathway. The Raf hit obtained also had atruncation of the kinase domain, creating a dominant negative form ofthe kinase. Other signaling molecules known to involve in TCR pathwaywere also discovered in our screen. They included PAG, CSK, SHP-1 andnucleolin.

PAK2 is a serine/threonine kinase and a member of the PAK family ofproteins. A cDNA encoding the Cdc24-binding domain of PAK2, but lackingthe kinase domain was isolated as a functional hit using the T cell CD69assay described herein. Another truncated form was also isolated usingthe same assay. Overexpression of this kinase domain-truncated form ofPAK2 (DN-PAK2), as well as the second mutant, in Jurkat T cells resultedin marked inhibition of TCR mediated CD69 upregulation. The inhibitoryeffect by overexpressing the DN-PAK2 was specific to T cells, since itfailed to affect the PCR-induced CD69 activation in BJAB cells.Introduction of the DN-PAK2 in primary T cells lead to inhibition ofIL-2 secretion following TCR and CD28 stimulation. In primary T cellsexpressing the DN-PAK2, the TCR-induced upregulation of CD40L wascompromised. Although PAK1 has been previously implicated inTCR-mediated signal transduction (see, e.g., Ku et al., EMBO J.20:457-465 (1998)), the data described herein show that the anti-PAK1antibody used in those studies cross-reacts with PAK2. Using TaqMan, thedata provided herein shows that PAK2 mRNA is much more abundant thanPAK1 in lymphoid cells and is abundantly expressed in humanhematopoietic cells. PAK2 is also involved in the TCR signaling pathway,as stimulation of TCR enhances PAK2 kinase activity, which peaked around5 minutes following the receptor ligation.

Function in primary T lymphocytes: The relevance of the CD69 screen hitsto physiological function of T cells was investigated in primary Tlymphocytes. The hit was subcloned into a retroviral vector under aconstitutively active promoter, followed by IRES-GFP. A protocol wasalso developed to couple successful retroviral infection to subsequenceT cell activation. Primary T lymphocytes are at the quiescent stage whenisolated from healthy donors. In order to be infected by retrovirus,primary lymphocytes need to be activated to progress in cell cycle.Fresh peripheral blood lymphocytes (PBL) contained typically T cells andB cells. The combined CD4+ and CD8+ cells represented total T cellpercentage, which was 81% in this particular donor. The remaining 19%CD4−CD8− cells were B cells as stained by CD19 (data not shown). Uponculturing on anti-CD3 and anti-CD28 coated dishes, primary T lymphocyteswere expanded and primary B cells and other cell types gradually diedoff in the culture. After infection, the culture contained virtually allT cells. Furthermore, primary T lymphocytes were successfully infectedby retroviruses. As seen with Jurkat cells (data not shown), GFPtranslated by way of IRES was not as abundant as GFP translated usingthe conventional Kozak sequence (comparing GFP geometric mean fromCRU5-IRES-GFP and CRU5-GFP). Nevertheless the percentage infectionremained similar. Insertion of a gene in front of IRES-GFP furtherreduced the expression level of GFP, which was observed with cell lines(data not shown) and here primary T lymphocytes. After allowing cells torest following infection, FACS sorted cells were divided into twopopulations: GFP− and GFP+. The sorted cells were immediately put intoculture. Anti-CD3 alone did not induce IL-2 production. This observationwas consistent with previous report on freshly isolated primary Tlymphocytes and confirmed the notion that prior culture and retroviralinfection did not damage the physiological properties of these primary Tlymphocytes. Addition of anti-CD28 in conjunction with anti-CD3 led torobust IL-2 production with vector-infected cells and the GFP−population of LckDN and PLCγ1DN-infected cells. The GFP+ cell populationfrom LckDN and PLCγ1DN-infected cells, however, were severed impaired inIL-2 production. As expect, the defect caused by LckDN and PLCγ1DN canbe completely rescued by stimulation using PMA and ionomycin. Takentogether, these results showed that Lck and PLCγ1 plays a role in IL-2production from primary T lymphocytes, consistently with theirinvolvement membrane proximal signaling events of T cell activation.These results also demonstrated a successful system to quickly validatehits from our functional genetic screens in primary cells.

Use of CD69 upregulation in drug screening: The discovery of importantimmune regulatory molecules from the T cell activation-induced CD69upregulation validated the relevance of this cell-based assay.Essentially such a cell-based assay offers the opportunity to discoverinhibitors of multiple targets such as Lck, ZAP70, PLCγ1 and PAK2. It isthe equivalent of multiplexing enzymatic assays with the additionaladvantage of cell permeability of compounds. It may even be possible toidentify novel compounds that block adaptor protein functions. Towardsthis end, the FACS assay of cell surface CD69 expression was convertedto a micro-titer plate based assay.

In conclusion, the strategy presented in this study demonstrates asuccessful approach to discover and validate important immune regulatorson a genome-wide scale. This approach, which requires no prior sequenceinformation, provides a tool for functional cloning of regulators innumerous signal transduction pathways. For example, B cellactivation-induced CD69 expression, IL-4-induced IgE class switch andTNF-induced NF-kB reporter gene expression are all amendable to thegenetic perturbation following introduction of retroviral cDNAlibraries. The outlined strategy is less biased compared to forcedintroduction of a handful of signaling molecules discovered in othercontext such as growth factor signal transduction. It also opens thedoor for discovering peptide inhibitors of immune modulatory proteins byscreening random peptide libraries expressed from the retroviral vector.

C. Methods

Cell culture: Human Jurkat T cells (clone N) were routinely cultured inRPMI 1640 medium supplemented with 10% fetal calf serum (Hyclone),penicillin and streptamycin. Phoenix A cells were grown in DMEMsupplemented with 10% fetal calf serum, penicillin and streptamycin. Toproduce the tTA-Jurkat cell line, Jurkat cells were infected with aretroviral construct which constitutively expresses the tetracyclinetransactivator protein and a reporter construct which expresses LyT2driven by a tetracycline responsive element (TRE). The tTA-Jurkat cellpopulation was optimized by sorting multiple sounds for highTRE-dependent expression of LyT2 in the absence of Dox and strongrepression of LyT2 expression in the presence Dox. The cells were alsosorted for maximal anti-TCR induced expression of CD69. Doxycycline wasused at a final concentration of 10 ng/ml for at least 6 days todownregulate expression of cDNAs from the TRE promoter.

Transfection and infection: Phoenix A packaging cells were transfectedwith retroviral vectors using calcium phosphate for 6 hours as standardprotocols. After 24 hours, supernatant was replaced with complete RPMImedium and virus was allowed to accumulate for an additional 24 hours.Viral supernatant was collected, filtered through a 0.2 μM filter andmixed with Jurkat cells at a density of 2.5×10⁵ cells/ml. Cells werespun at room temperature for 3 hours at 3000 rpm, followed by overnightincubation at 37° C. Transfection and infection efficiencies weremonitored by GFP expression and functional analysis was carried out 2-4days after infection.

Libraries: RNA extracted from human lymph node, thymus, spleen and bonemarrow was used to produce two cDNA libraries; one random primed anddirectionally cloned and the second non-directionally cloned andprovided with 3 exogenous ATG in 3 frames. cDNAs were cloned into thepTRA-exs vector giving robust doxycycline-regulable transcription ofcDNAs from the TRE promoter. The total combined library complexity was5×10⁷ independent clones.

Stimulation: For CD69 upregulation experiments, tTA-Jurkat cells weresplit to 2.5×10⁵ cells/ml 24 hours prior to stimulation. Cells were spunand resuspended at 5×10⁵ cells/ml in fresh complete RPMI medium in thepresence of 100 ng/ml C305 (anti-Jurkat clonotypic TCR) or 5 ng/ml PMAhybridoma supernatant for 20-26 hours at 37° C., and then assayed forsurface CD69 expression.

Cell surface marker analysis: Jurkat-N cells were stained with anAPC-conjugated mouse monoclonal anti-human CD69 antibody (Caltag) at 4°C. for 20 minutes and analyzed using a Facscalibur instrument (BectonDickinson) with Cellquest software. Cell sorts were performed on a MoFlo(Cytomation).

cDNA screen: Phoenix A packaging cells were transfected with a mixtureof the two tTA regulated retroviral pTRA-exs cDNA libraries. Supernatantcontaining packaged viral particles was used to infect tTA-Jurkat cellswith an efficiency of ˜85%. After 4 days of cDNA expression, libraryinfected cells were stimulated with 0.3 μg/ml C305 for 20-26 hours,stained with APC-conjugated anti-CD69, and lowest CD69-expressing cellsstill expressing CD3 (CD69^(low)CD3⁺) were isolated using a fluorescenceactivated cell sorter. Sorting was repeated over multiple rounds with a6-day rest period between stimulations until the population wassignificantly enriched for non-responders. Single cells were depositedfrom 4 separate rounds of sorting. Cell clones were expanded in thepresence and absence of Dox, stimulated and analyzed for CD69upregulation.

Isolation of cDNA inserts: PCR primers were designed to amplify cDNAinserts from both libraries and did not amplify Lyt2 that was also underTRE regulation. The primers used contained flanking BstXI sites forsubsequent cloning to pTRA-IRES-GFP vector. RT-PCR cloning was achievedwith kits from Clontech or Life Technologies. The gel-purified RT-PCRproducts were submitted for sequencing directly and simultaneouslydigested for subcloning. Dominant negative ZAP70 (KI) and ZAP70SH2 (N+C)as well as selected hits from cDNA screens were subcloned to theretroviral pTRA-IRES-GFP vector. Selected hits form cDNA screens werealso subcloned to CRU5-IRES-GFP for infection of human primary Tlymphocytes and examination of IL-2 production.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-28. (canceled)
 29. A method for identifying a compound that modulatesT lymphocyte activation, the method comprising the steps of: (i)contacting the compound with a PAK2 polypeptide or an active fragmentthereof, the PAK2 polypeptide or fragment thereof comprising apolypeptide having at least 90% identity to an amino acid sequence ofSEQ ID NO:2; (ii) determining the effect of the compound upon the PAK2polypeptide or fragment thereof in vitro using a kinase assay, therebyidentifying a compound that modulates T lymphocyte activation.
 30. Themethod of claim 29, wherein the PAK2 polypeptide comprises an amino acidsequence of SEQ ID NO:2.
 31. The method of claim 29, wherein the PAK2polypeptide is encoded by a nucleic acid comprising a nucleotidesequence of SEQ ID NO:1.
 32. The method of claim 29, wherein thepolypeptide is recombinant.
 33. The method of claim 29, wherein the PAK2polypeptide is attached to a sold phase substrate.
 34. The method ofclaim 29, wherein the compound is an antibody.
 35. The method of claim29, wherein the compound is an antisense molecule.
 36. The method ofclaim 29, wherein the compound is a small organic molecule.
 37. Themethod of claim 29, wherein the compound is a peptide
 38. The method ofclaim 37, wherein the peptide is circular.
 39. The method of claim 37,wherein the peptide is a fragment of the PAK2 kinase domain.